Kinetics Of Open Complex Formation Research Articles

Cooperative stabilization of Mycobacterium tuberculosis rrnAP3 promoter open complexes by RbpA and CarD.

The essential mycobacterial transcriptional regulators RbpA and CarD act to modulate transcription by associating to the initiation complex and increasing the flux of transcript production. Each of these factors interacts directly with the promoter DNA template and with RNA polymerase (RNAP) holoenzyme. We recently reported on the energetics of CarD-mediated open complex stabilization on the Mycobacterium tuberculosis rrnAP3 ribosomal promoter using a stopped-flow fluorescence assay. Here, we apply this approach to RbpA and show that RbpA stabilizes RNAP-promoter open complexes (RPo) via a distinct mechanism from that of CarD. Furthermore, concentration-dependent stopped-flow experiments with both factors reveal positive linkage (cooperativity) between RbpA and CarD with regard to their ability to stabilize RPo. The observation of positive linkage between RbpA and CarD demonstrates that the two factors can act on the same transcription initiation complex simultaneously. Lastly, with both factors present, the kinetics of open complex formation is significantly faster than in the presence of either factor alone and approaches that of E. coli RNAP on the same promoter. This work provides a quantitative framework for the molecular mechanisms of these two essential transcription factors and the critical roles they play in the biology and pathology of mycobacteria.

Kinetics and Mechanism of Formation and Stabilization of the RNA Polymerase-Promoter Open Compex

Specific binding of E. coli RNA polymerase holoenzyme (RNAP; α2ββ’σ70) to promoter DNA sets in motion a series of conformational changes that advance the initial closed complex (RPc), open 12-14 bp including the −10 region and the transcription start site, and at some promoters then stabilize the initial open complex dramatically. Our research focuses on the kinetics and mechanism of these conformational changes at different promoters, using Forster resonance energy transfer (FRET) and protein induced fluorescence enhancement (PIFE) together with fast footprinting and filter binding assays to determine the kinetics of open complex formation and dissociation, and to obtain structural and thermodynamic information about key intermediates. FRET and PIFE results to date demonstrate the importance of wrapping of upstream promoter DNA on RNAP for efficient bending of the downstream duplex into the active site cleft to form the advanced closed complex which is opened by RNAP in the rate limiting step. The initial open complex is found to be unstable, and is subsequently stabilized at some but not all promoters by conformational changes and interactions of in-cleft and downstream mobile elements of RNAP, directed by the discriminator sequence of the promoter. Dissociation kinetic studies with PIFE, FRET and filter binding assays are being used to probe the mechanism of these conformational changes in open complex stabilization. This project is funded by NIH support(GM103061).

CarD stabilizes mycobacterial open complexes via a two-tiered kinetic mechanism.

CarD is an essential and global transcriptional regulator in mycobacteria. While its biological role is unclear, CarD functions by interacting directly with RNA polymerase (RNAP) holoenzyme promoter complexes. Here, using a fluorescent reporter of open complex, we quantitate RPo formation in real time and show that Mycobacterium tuberculosis CarD has a dramatic effect on the energetics of RNAP bound complexes on the M. tuberculosis rrnAP3 ribosomal RNA promoter. The data reveal that Mycobacterium bovis RNAP exhibits an unstable RPo that is stabilized by CarD and suggest that CarD uses a two-tiered, concentration-dependent mechanism by associating with open and closed complexes with different affinities. Specifically, the kinetics of open-complex formation can be explained by a model where, at saturating concentrations of CarD, the rate of bubble collapse is slowed and the rate of opening is accelerated. The kinetics and open-complex stabilities of CarD mutants further clarify the roles played by the key residues W85, K90 and R25 previously shown to affect CarD-dependent gene regulation in vivo. In contrast to M. bovis RNAP, Escherichia coli RNAP efficiently forms RPo on rrnAP3, suggesting an important difference between the polymerases themselves and highlighting how transcriptional machinery can vary across bacterial genera.

Effects of distortions by A-tracts of promoter B-DNA spacer region on the kinetics of open complex formation by Escherichia coli RNA polymerase.

A-tracts in DNA due to their structural morphology distinctly different from the canonical B-DNA form play an important role in specific recognition of bacterial upstream promoter elements by the carboxyl terminal domain of RNA polymerase alpha subunit and, in turn, in the process of transcription initiation. They are only rarely found in the spacer promoter regions separating the -35 and -10 recognition hexamers. At present, the nature of the protein-DNA contacts formed between RNA polymerase and promoter DNA in transcription initiation can only be inferred from low resolution structural data and mutational and crosslinking experiments. To probe these contacts further, we constructed derivatives of a model Pa promoter bearing in the spacer region one or two An (n = 5 or 6) tracts, in phase with the DNA helical repeat, and studied the effects of thereby induced perturbation of promoter DNA structure on the kinetics of open complex (RPo) formation in vitro by Escherichia coli RNA polymerase. We found that the overall second-order rate constant ka of RPo formation, relative to that at the control promoter, was strongly reduced by one to two orders of magnitude only when the A-tracts were located in the nontemplate strand. A particularly strong 30-fold down effect on ka was exerted by nontemplate A-tracts in the -10 extended promoter region, where an involvement of nontemplate TG (-14, -15) sequence in a specific interaction with region 3 of sigma-subunit is postulated. A-tracts in the latter location caused also 3-fold slower isomerization of the first closed transcription complex into the intermediate one that precedes formation of RPo, and led to two-fold faster dissociation of the latter. All these findings are discussed in relation to recent structural and kinetic models of RPo formation.

Effects of distortions by A-tracts of promoter B-DNA spacer region on the kinetics of open complex formation by Escherichia coli RNA polymerase.

A-tracts in DNA due to their structural morphology distinctly different from the canonical B-DNA form play an important role in specific recognition of bacterial upstream promoter elements by the carboxyl terminal domain of RNA polymerase alpha subunit and, in turn, in the process of transcription initiation. They are only rarely found in the spacer promoter regions separating the -35 and -10 recognition hexamers. At present, the nature of the protein-DNA contacts formed between RNA polymerase and promoter DNA in transcription initiation can only be inferred from low resolution structural data and mutational and crosslinking experiments. To probe these contacts further, we constructed derivatives of a model Pa promoter bearing in the spacer region one or two An (n = 5 or 6) tracts, in phase with the DNA helical repeat, and studied the effects of thereby induced perturbation of promoter DNA structure on the kinetics of open complex (RPo) formation in vitro by Escherichia coli RNA polymerase. We found that the overall second-order rate constant ka of RPo formation, relative to that at the control promoter, was strongly reduced by one to two orders of magnitude only when the A-tracts were located in the nontemplate strand. A particularly strong 30-fold down effect on ka was exerted by nontemplate A-tracts in the -10 extended promoter region, where an involvement of nontemplate TG (-14, -15) sequence in a specific interaction with region 3 of sigma-subunit is postulated. A-tracts in the latter location caused also 3-fold slower isomerization of the first closed transcription complex into the intermediate one that precedes formation of RPo, and led to two-fold faster dissociation of the latter. All these findings are discussed in relation to recent structural and kinetic models of RPo formation.

Kinetics of Transcription Initiation at lacP1: MULTIPLE ROLES OF CYCLIC AMP RECEPTOR PROTEIN

The cyclic AMP receptor protein (CRP) acts as a transcription activator at many promoters of Escherichia coli. We have examined the kinetics of open complex formation at the lacP1 promoter using tryptophan fluorescence of RNA polymerase and DNA fragments with 2-aminopurine substituted at specific positions. Apart from the closed complex formation and promoter clearance, we were able to detect three steps. The first step after the closed complex formation leads to a rapid increase of 2-aminopurine fluorescence. This was followed by another rapid step in which quenching of tryptophan fluorescence of RNA polymerase was observed. The slowest step detected by 2-aminopurine fluorescence increase is assigned to the final open complex formation. We have found that CRP not only enhances RNA polymerase binding at the promoter, but also enhances the slowest isomerization step by about 2-fold. Furthermore, potassium permanganate probing shows that the conformation of the open complex in the presence of CRP appears qualitatively and quantitatively different from that in the absence of CRP, suggesting that contact with RNA polymerase is maintained throughout the transcription initiation.

Effect of A(n) tracts within the UP element proximal subsite of a model promoter on kinetics of open complex formation by Escherichia coli RNA polymerase.

In the open transcription complex (RPo), Escherichia coli RNA polymerase sigma(70) and alpha subunits are known to be in contact with each other and with the promoter region overlapping the -35 hexamer and the proximal part of the UP element. To probe the effect of A(n) DNA bending tracts in this region on initiation of transcription, kinetics of the formation of RPo by Escherichia coli RNA polymerase at two groups of synthetic consensus-like promoters bearing single DNA bending tracts (i). A(5 )within the proximal subsite region of the UP element (promoters Pk and Pl) and (ii). A(5)(Pg) or A(8)(Pm) in the region including the downstream end of the proximal UP subsite and the -35 consensus hexamer was studied in vitro using the fluorescence-detected abortive initiation assay. The kinetic data obtained demonstrate that the overall second-order rate constant k(a) of RPo formation is: (i.by almost one order of magnitude larger at Pk and Pl, relative to that at a control unbent promoter, and mainly due to a higher value of the equilibrium constant, K(1), of the initial closed complex; and (ii). several-fold smaller at Pg and Pm owing to a strongly decreased value of K(1). For Pm, the latter parameter was found to be dependent exponentially on four Mg(2+) ions, as compared with the seven ions remaining in equilibrium with the initial closed complex at the parent Pa promoter. This indicates that promoter region bearing a stiff A(8).T(8) fragment of B -DNA forms a smaller number of ionic contacts with the alpha subunit. These findings provide a new insight to and support the present model of interactions between RNA polymerase alpha and sigma(70) subunits with the proximal UP subsite and the -35 region of promoters.

Effect of A(n) tracts within the UP element proximal subsite of a model promoter on kinetics of open complex formation by Escherichia coli RNA polymerase.

In the open transcription complex (RPo), Escherichia coli RNA polymerase sigma(70) and alpha subunits are known to be in contact with each other and with the promoter region overlapping the -35 hexamer and the proximal part of the UP element. To probe the effect of A(n) DNA bending tracts in this region on initiation of transcription, kinetics of the formation of RPo by Escherichia coli RNA polymerase at two groups of synthetic consensus-like promoters bearing single DNA bending tracts (i). A(5 )within the proximal subsite region of the UP element (promoters Pk and Pl) and (ii). A(5)(Pg) or A(8)(Pm) in the region including the downstream end of the proximal UP subsite and the -35 consensus hexamer was studied in vitro using the fluorescence-detected abortive initiation assay. The kinetic data obtained demonstrate that the overall second-order rate constant k(a) of RPo formation is: (i.by almost one order of magnitude larger at Pk and Pl, relative to that at a control unbent promoter, and mainly due to a higher value of the equilibrium constant, K(1), of the initial closed complex; and (ii). several-fold smaller at Pg and Pm owing to a strongly decreased value of K(1). For Pm, the latter parameter was found to be dependent exponentially on four Mg(2+) ions, as compared with the seven ions remaining in equilibrium with the initial closed complex at the parent Pa promoter. This indicates that promoter region bearing a stiff A(8).T(8) fragment of B -DNA forms a smaller number of ionic contacts with the alpha subunit. These findings provide a new insight to and support the present model of interactions between RNA polymerase alpha and sigma(70) subunits with the proximal UP subsite and the -35 region of promoters.

Different Roles for Basic and Aromatic Amino Acids in Conserved Region 2 of Escherichia coli ς70 in the Nucleation and Maintenance of the Single-stranded DNA Bubble in Open RNA Polymerase-Promoter Complexes

Amino acid residues in region 2 of final sigma(70) have been shown to play an important role in the strand separation step that is necessary for formation of the functional or open RNA polymerase-promoter complex. Here we present a comparison of the roles of basic and aromatic amino acids in the accomplishment of this process, using RNA polymerase bearing alanine substitutions for both types of amino acids in region 2. We determined the effects of the substitutions on the kinetics of open complex formation, as well as on the ability of the RNA polymerase to form complexes with single-stranded DNA, and with forked DNA duplexes carrying a single-stranded overhang consisting of bases in the -10 region. We concluded that two basic amino acids (Lys(414) and Lys(418)) are important for promoter binding and demonstrated distinct roles, at a subsequent step, for two aromatic amino acids (Tyr(430) and Trp(433)). It is likely that these four amino acids, which are close to each other in the structure of final sigma(70), together are involved in the nucleation of the strand separation process.

Peculiar 2-Aminopurine Fluorescence Monitors the Dynamics of Open Complex Formation by Bacteriophage T7 RNA Polymerase

The kinetics of promoter binding and open complex formation in bacteriophage T7 RNA polymerase was investigated using 2-aminopurine (2-AP) modified promoters. 2-AP serves as an ideal probe to measure the kinetics of open complex formation because its fluorescence is sensitive to both base-unpairing and base-unstacking and to the nature of the neighboring bases. All four 2-AP bases in the TATA box showed an increase in fluorescence with similar kinetics upon binding to the T7 RNA polymerase, indicating that the TATA sequence becomes unpaired in a concerted manner. The 2-AP at -4 showed a peculiarly large increase in fluorescence upon binding to the T7 RNA polymerase. Based on the recent crystal structure of the T7 RNA polymerase-DNA complex, we propose that the large fluorescence increase is due to unstacking of the 2-AP base at -4 from the guanine at -5, during open complex formation. The unstacking may be a critical event in directing and placing the template strand correctly in the T7 RNA polymerase active site upon promoter melting for template directed RNA synthesis. Based on equilibrium fluorescence and stopped-flow kinetic studies, we propose that a fast form of T7 RNA polymerase binds promoter double-stranded DNA by a three-step mechanism. The initial collision complex or a closed complex, ED(c) is formed with a K(d) of 1.8 microm. This complex isomerizes to an open complex, ED(o1), in an energetically unfavorable reaction with an equilibrium constant of 0.12. The ED(o1) further isomerizes to a more stable open complex, ED(o2), with a rate constant around 300 s(-1). Thus, in the absence of the initiating nucleotide, GTP, the overall equilibrium constant for closed to open complex conversion is 0.5 and the net rate of open complex formation is nearly 150 s(-1).

A Real-Time Fluorescence Method to Monitor the Melting of Duplex DNA during Transcription Initiation by RNA Polymerase

The melting of duplex DNA in the vicinity of the transcription start site is an essential step of transcription initiation. Here we describe a fluorescent promoter technique which allows the melting of promoter DNA to be observed in a real-time manner with high sensitivity. We have constructed a 114-bplacUV5 promoter fragment (−89 to +25) which contains a fluorescence probe in the region between the −10 consensus hexamer and the transcription start site. This region was chosen to incorporate a fluorescence probe as it undergoes strand separation subsequent to binding RNA polymerase (RNAP) (i.e., open complex formation). Upon mixing RNAP and fluorochrome-labeled promoter a time-dependent biphasic change in fluorescence was observed. The second slower component was shown to be due to the open complex by comparing the fluorescence data with the kinetics of open complex formation as measured by using alternative methods of open complex detection. The rate constants for open complex formation and dissociation were determined and found to be in excellent agreement with previously reported values. The techniques presented herein can generally be applied to other systems. Furthermore, this method will serve as an important research tool as well as it could be used in designing high-throughput assays involving transcription complexes.

General Method of Analysis of Kinetic Equations for Multistep Reversible Mechanisms in the Single-Exponential Regime: Application to Kinetics of Open Complex Formation between E σ70 RNA Polymerase and λP R Promoter DNA

A novel analytical method based on the exact solution of equations of kinetics of unbranched first- and pseudofirst-order mechanisms is developed for application to the process of E σ 70 RNA polymerase (R)– λP R promoter (P) open complex formation, which is described by the minimal three-step mechanism with two kinetically significant intermediates (I 1, I 2), {formula not available us MathML} where the final product is an open complex RP o. The kinetics of reversible and irreversible association (pseudofirst order, [R] ≫ [P]) to form long-lived complexes (RP o and I 2) and the kinetics of dissociation of long-lived complexes both exhibit single exponential behavior. In this situation, the analytical method provides explicit expressions relating observed rate constants to the microscopic rate constants of mechanism steps without use of rapid equilibrium or steady-state approximations, and thereby provides a basis for interpreting the composite rate constants of association ( k a), isomerization ( k i), and dissociation ( k d) obtained from experiment for this or any other sequential mechanism of any number of steps. In subsequent papers, we apply this formalism to analyze kinetic data obtained in the reversible and irreversible binding regimes of E σ 70 RNA polymerase (R)– λP R promoter (P) open complex formation.

The differential effects of guanosine tetraphosphate on open complex formation at the Escherichia coli ribosomal protein promoters rplJ and rpsA P1

The effects of guanosine tetraphosphate (ppGpp) on inhibition of single-round in vitro transcription and on the kinetics of open complex formation were investigated at the Escherichia coli ribosomal protein promoters rplJ and rpsA P1. The two promoters differ in their saturation characteristics and sensitivities to ppGpp. With a 10:1 molar ratio of RNA polymerase (RNAP) to DNA, saturation of transcription activity and weak inhibition (≈30%) are observed at rplJ, in contrast to the weak activity and strong inhibition (≈80%) at rpsA P1. In the absence of ppGpp, the two promoters show a threefold difference in the overall rate constants of association ( k a) (6.5×10 7 M −1 s −1 at rplJ and 2.0×10 7 M −1 s −1 at rpsA P1), while the dissociation rate constants ( k d) are similar (≈4.8×10 −5 s −1). The addition of ppGpp causes a twofold reduction in k 2 (isomerisation constant) at rplJ and a threefold decrease in K B (equilibrium constant of RNAP binding) at rpsA P1. There is a significant twofold increase in k d at rplJ, compared with smaller changes at rpsA P1 and at the non-stringent lacUV5 promoter. These results indicate that ppGpp affects the formation and stability of the open complex at the rplJ promoter, in contrast to the inhibition of RNAP binding to the rpsA P1 promoter.

Obligatory activator-polymerase addition order at promoters

The kinetics of open complex formation were measured by migration retardation assay and DNase I footprinting at the activator-dependent promoters ara P1, lac P1 and gal P1. In each case, the rate of open complex formation was significantly faster if the activator, AraC for ara and CAP for lac and gal, had been added before RNA polymerase. The results indicate that complexes of transcriptional activators, RNA polymerase and promoter can exist in two states, one which can form open complexes rapidly and one which cannot.

Regulation of open complex formation at the Escherichia coli galactose operon promoters : Simultaneous interaction of RNA polymerase, gal repressor and CAP/cAMP

The regulation of open complex formation at the Escherichia coli galactose operon promoters by galactose repressor and catabolite activator protein/cyclic AMP (CAP/cAMP) was investigated in DNA-binding and kinetic experiments performed in vitro. We found that gal repressor and CAP/cAMP bind to the gal regulatory region independently, resulting in simultaneous occupancy of the two gal operators and the CAP/cAMP binding site. Both CAP/cAMP and gal repressor altered the partitioning of RNA polymerase between the two overlapping gal promoters. Open complexes formed in the absence of added regulatory proteins were partitioned between gal P1 and P2 with occupancies of 25% and 75%, respectively. CAP/cAMP caused open complexes to be formed nearly exclusively at P1 (98% occupancy), gal repressor caused a co-ordinated, but incomplete, switch in promoter partitioning from P1 to P2 in both the absence and presence of . We measured the kinetic constants governing open complex formation and decay at the gal promoters in the absence and presence of gal repressor and CAP/cAMP. had the largest effect on the kinetics of open complex formation, resulting in a 30-fold increase in the apparent binding constant. We conclude that the regulation of open complex formation at the gal promoters does not result from competition between gal repressor, and RNA polymerase for binding at the gal operon regulatory region, but instead results from the interactions of the three proteins during the formation of a nucleoprotein complex on the gal DNA fragment. Finally, we present a kinetic model for the regulation of open complex formation at the gal operon.

Overlapping promoters and their control in Escherichia coli: the gal case.

Two overlapping promoters compete for RNA polymerase in the region that controls the expression of the galactose operon in Escherichia coli. Kinetics of open complex formation at P1 and P2 can be followed through the rate of formation of two specific abortive transcripts. The corresponding forward kinetic constants appear to be identical over a wide range of enzyme concentrations and temperatures, indicating that the two processes are strongly coupled. We propose a scheme accounting for our observations. In a first step, the competition between the two sites is a simple kinetic process, involving the "on" rate constants. In a second step, a slow reequilibration occurs, implicating the "off" rate constants and the conversion of one open complex to the other through a set of closed complexes. The first step is clearly affected when the complex between cyclic AMP and its receptor is bound at the activator site. An estimate of the various rate constants describing open complex formation at P1 and P2 is provided, as well as a qualitative description of the effect of the activator complex on these two pathways.

Kinetics of open complex formation between Escherichia coli RNA polymerase and the lac UV5 promoter. Evidence for a sequential mechanism involving three steps.

The forward and reverse kinetics of open complex formation between Escherichia coli RNA polymerase and the lac UV5 promoter have been studied in the temperature range of 15-42 degrees C. The standard two-step model, involving the formation of a closed intermediate, RPc, followed by an isomerization that leads to the active complex RPo, could not account for the present data. The promoter-enzyme lifetime measurements showed an inverse temperature dependence (apparent activation energy, -35 kcal/mol). A third step, which is very temperature dependent and which is very rapid at 37 degrees C, was postulated to involve the unstacking of DNA base pairs that immediately precedes open complex formation. Evidence for incorporating a new binary complex, RPi, in the pathway was provided by experiments that distinguished between stably bound species and active promoter after temperature-jump perturbations. These experiments allowed measurement of the rate of reequilibration between the stably bound species and determination of the corresponding equilibrium constant. They indicated that the third step became rate limiting below 20 degrees C; this prediction was checked by an analysis of the forward kinetics. A quantitative evaluation of the parameters involved in this three-step model is provided. Similar experiments were performed on a negatively supercoiled template: in this case the third equilibrium was driven toward formation of the open complex even at low temperature, and the corresponding step was not rate limiting.